CN109294554B - Fluorescent microsphere with carboxyl-rich surface and synthesis method thereof - Google Patents
Fluorescent microsphere with carboxyl-rich surface and synthesis method thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent microsphere with carboxyl-rich surface and a synthesis method thereof. The surface of the microsphere is rich in carboxyl groups and is hybridized by rare earth coordination fluorescent molecules, and the particle size of the microsphere is 0.1-2 mu m. The invention has better controllability and higher repeatability, and the prepared microsphere has uniform particle size, controllable size and high fluorescence efficiency, is stable to disperse in a larger pH range and can be stably dispersed in solvents such as water, ethanol, acetone, acetonitrile and the like; the required raw materials are nontoxic, environment-friendly, low in price, simple to operate and low in equipment requirement. The invention can be widely applied to the fields of chemistry, biology, material science and the like; considering that the microsphere prepared by the invention has higher surface carboxyl density compared with common carboxylated microspheres, more proteins, DNA or other molecules with amino terminal can be fixed on the microsphere, and the microsphere has wide application prospect in the biomedical field, especially in the aspects of fluorescence imaging, cancer radiation therapy, fluorescence labeling, fluorescence analysis and the like.
Description
Technical Field
The invention relates to a fluorescent microsphere with carboxyl-rich surface and a synthesis method thereof, and relates to the technical field of high polymer materials and synthesis thereof.
Background
The fluorescent microsphere generally refers to a microsphere with fluorescent substances marked on the surface of the microsphere or the internal structure of the microsphere containing the fluorescent substances, and the microsphere can excite fluorescence by the stimulation of external energy, and the shape of the microsphere is generally spherical; fluorescent microspheres can be classified into two types according to the carrier and fluorescent substance: one is inorganic/organic fluorescent microsphere, and the other is organic/organic microsphere; the organic/organic microsphere can conveniently modify various functional groups in actual demands on the surface of the microsphere, different comonomers and polymerization processes can be selected to design the polymer microsphere, and the biological macromolecules fixed on the surface of the microsphere can perform various reactions in homogeneous phase due to the fact that the density of the microsphere is similar to that of water.
The method for preparing the fluorescent microsphere commonly comprises a physical adsorption method, a self-assembly method, an embedding method, a chemical bond and method, a copolymerization method and the like, but in the specific synthesis steps, the existing synthesis method is poor in stability, the prepared microsphere particle size is relatively uncontrollable, the synthesis method is complex, the price and the cost are high, and further improvement is needed.
Disclosure of Invention
The invention aims at overcoming the defects, and aims to provide the fluorescent microsphere with the carboxyl-rich surface, which has uniform particle size, controllable size and high fluorescence efficiency, and the synthesis method thereof.
The technical scheme adopted by the invention is as follows: the surface of the fluorescent microsphere is rich in carboxyl, the surface of the microsphere is hybridized by rare earth coordination fluorescent molecules, and the particle size of the microsphere is 0.1-2 mu m.
The synthesis method of the fluorescent microsphere with the surface rich in carboxyl comprises the following steps:
1) Synthesizing high molecular microsphere with carboxyl-rich surface by soap-free emulsion copolymerization reaction: dissolving a polymer monomer for polymerization and olefine acid in methanol, taking sodium p-styrenesulfonate as a copolymerization emulsifier, adding an initiator, reacting under the protection of nitrogen, centrifuging, washing, and then dispersing in distilled water again;
2) Preparing fluorescent microspheres with carboxyl-rich surfaces: regulating the pH value of the mixed solution in the prepared high polymer microsphere aqueous dispersion liquid with the surface rich in carboxyl; then adding rare earth (III) ion solution for reaction, centrifuging, washing and vacuum drying after the reaction is finished; dispersing the obtained dry microspheres in ethanol, adding an organic ligand solution for reaction, centrifuging, washing and drying in vacuum after the reaction is finished to obtain fluorescent microspheres with surfaces rich in carboxyl groups.
The polymer monomer for polymerization is at least one selected from styrene, substituted styrene, methacrylate and acrylic ester.
The olefine acid is at least one selected from itaconic acid, fumaric acid, itaconic anhydride, fumaric anhydride, unsaturated di-or poly-carboxylic acid and corresponding anhydride.
Doping polyvinylpyrrolidone into the copolymerization emulsifier;
further, the polyvinylpyrrolidone is at least one of PVP K-30, PVP 360 and PVP 106.
The initiator for polymerization is at least one selected from potassium persulfate, azodiisobutyronitrile and benzoyl peroxide.
In the step 1), the dosage of the olefine acid is 0.5-10% of that of the polymerized monomer, the dosage of the copolymerized emulsifier is 0.2-2% of that of the polymerized monomer, and the dosage of the initiator is 1-5% of that of the polymerized monomer according to mass percent;
the reaction temperature is 40-90 ℃ under the protection of nitrogen, and the reaction time is 12-48 hours.
In the step 2), the amine used for regulating the pH value of the mixed solution is at least one selected from triethylamine and ethylenediamine, and the rare earth is at least one selected from terbium, europium, samarium, dysprosium and gadolinium; the pH value is 5-10, the reaction temperature is 30-85 ℃, and the reaction time is 3-48 hours.
In the step 2), the organic ligand is selected from at least one of 2,2' -bipyridine, 1, 10-phenanthroline, 8-hydroxyquinoline, indole, beta-diketone, aromatic carboxylic acid, triphenylphosphine oxide, dialkyl sulfoxide, pyridine nitrogen oxide, quinoline nitrogen oxide, macrocyclic polyether, macrocyclic polyketone, porphyrin, phthalocyanine, polyene compound and the like and derivatives thereof in the heterocyclic compound.
In the step 2), the amount of amine is 0.5-10% of carboxylated microspheres, the amount of rare earth (III) ions is 0.5-5% of carboxylated microspheres, the amount of organic ligand is 0.5-10% of carboxylated microspheres, and the balance is ethanol and water.
The invention has the advantages that: the invention has better controllability and higher repeatability, and the prepared microsphere has uniform particle size, controllable size and high fluorescence efficiency, is stable to disperse in a larger pH range and can be stably dispersed in solvents such as water, ethanol, acetone, acetonitrile and the like; the required raw materials are nontoxic, environment-friendly, low in price, simple to operate and low in equipment requirement. The invention can be widely applied to the fields of chemistry, biology, material science and the like; considering that the microsphere prepared by the invention has higher surface carboxyl density compared with common carboxylated microspheres, more proteins, DNA or other molecules with amino terminal can be fixed on the microsphere, and the microsphere has wide application prospect in the biomedical field, especially in the aspects of fluorescence imaging, cancer radiation therapy, fluorescence labeling, fluorescence analysis and the like.
Drawings
FIG. 1 is a TEM image of a polymer microsphere poly (St-co-IA) rich in carboxyl groups on the surface of the polymer microsphere.
FIG. 2 is a TEM image of carboxyl-enriched fluorescent microspheres of the present invention poly (St-co-IA)/Tb/phen.
FIG. 3 shows the fluorescence emission spectrum (detection wavelength: 295 nm) of carboxyl group-rich fluorescent microspheres poly (St-co-IA)/Tb/phen) of the present invention.
FIG. 4 fluorescence emission spectrum (detection wavelength: 295 nm) of carboxyl group-enriched fluorescent microsphere poly (St-co-IA)/Eu/phen) of the present invention.
FIG. 5 is a digital photograph of a carboxyl-rich fluorescent microsphere poly (St-co-IA)/Tb/phen) film of the present invention under 254 nm ultraviolet light irradiation.
FIG. 6 is a digital photograph of a fluorescent microsphere poly (St-co-IA)/Eu/phen) film rich in carboxyl groups on the surface of the present invention under 254 nm ultraviolet light irradiation.
FIG. 7 is a digital photograph of 254 nm UV light of an aqueous dispersion of carboxyl-rich fluorescent microspheres poly (St-co-IA)/Tb/phen) of the present invention.
FIG. 8 is a digital photograph of an aqueous dispersion of carboxyl-enriched fluorescent microspheres poly (St-co-IA)/Eu/phen) of the present invention under 254 nm ultraviolet light.
Detailed Description
Embodiment one:
1) Preparing carboxylated microspheres:
a four-necked round bottom flask equipped with an air duct, reflux condenser and feed port was placed in an oil bath heater and mechanical stirring with a polytetrafluoroethylene stirring rod was started and the stirring speed was maintained at 400 rpm. Distilled water, methanol, styrene, sodium p-styrenesulfonate and itaconic acid are sequentially added, the reaction system is closed, and nitrogen is introduced under stirring to remove the original oxygen in the system. After the temperature was raised to 70℃C, a potassium persulfate solution was added to the reaction mixture to start the reaction, and the reaction was stopped after stirring at a constant temperature for 24 hours. The emulsion collected was the desired poly (St-co-IA) microspheres. Unreacted IA and other reagents were removed by high-speed centrifugation, and the microspheres obtained by centrifugation were then redispersed in distilled water.
2) Preparing carboxylated fluorescent microspheres:
dispersing the poly (St-co-IA) microspheres prepared above into an aqueous solution, and adjusting the pH value of the mixed solution by adding a certain amount of triethylamine; then, terbium (III) ion solution was slowly added with stirring, and the system temperature was kept at 55℃throughout the reaction. After the reaction was completed, the microspheres were collected by high-speed centrifugation, washed several times with water and ethanol to remove unreacted terbium (iii) ions and other reagents (solvents), and then dried in vacuo at 50 ℃.
Dispersing poly (St-co-IA)/Tb prepared in the ethanol solution, slowly adding the phenanthroline ethanol solution under stirring, and keeping the system temperature at 40 ℃ in the whole reaction. After the reaction, the microspheres are collected by high-speed centrifugation, washed with water and ethanol for several times to remove unreacted phenanthroline and other reagents (solvents), and then dried in vacuum at 50 ℃.
Embodiment two: 1) Preparing carboxylated microspheres:
a four-necked round bottom flask equipped with an air duct, reflux condenser and feed port was placed in an oil bath heater and mechanical stirring with a polytetrafluoroethylene stirring rod was started and the stirring speed was maintained at 400 rpm. Distilled water, methanol and methacrylic acid esters are sequentially added, the reaction system is closed, and nitrogen is introduced under stirring to remove the original oxygen in the system. After the temperature was raised to 60 ℃, a potassium persulfate solution was added to the reaction mixture to start the reaction, and the reaction was stopped after stirring at a constant temperature for 30 hours. The collected emulsion is the desired microsphere. Unreacted IA and other reagents were removed by high-speed centrifugation, and the microspheres obtained by centrifugation were then redispersed in distilled water.
2) Preparing carboxylated fluorescent microspheres:
dispersing the microspheres prepared in the water solution, and adjusting the pH value of the mixed solution by adding a certain amount of ethylenediamine; then, terbium (III) ion solution was slowly added with stirring, and the system temperature was maintained at 60℃throughout the reaction. After the reaction was completed, the microspheres were collected by high-speed centrifugation, washed several times with water and ethanol to remove unreacted terbium (iii) ions and other reagents (solvents), and then dried in vacuo at 50 ℃.
Dispersing the prepared solution in ethanol solution, slowly adding pyridine nitrogen oxide solution under stirring, and maintaining the system temperature at 40 ℃ during the whole reaction. After the reaction was completed, the microspheres were collected by high-speed centrifugation, washed several times with water and ethanol to remove unreacted pyridine nitrogen oxides and other reagents (solvents), and then dried in vacuo at 50 ℃.
Embodiment III:
1) Preparing carboxylated microspheres:
a four-necked round bottom flask equipped with an air duct, reflux condenser and feed port was placed in an oil bath heater and mechanical stirring with a polytetrafluoroethylene stirring rod was started and the stirring speed was maintained at 400 rpm. Distilled water, methanol and acrylic ester are sequentially added, sodium styrene sulfonate and fumaric acid are added, the reaction system is closed, and nitrogen is introduced under stirring to remove the original oxygen in the system. After the temperature was raised to 70C, the reaction was started by adding azobisisobutyronitrile solution to the reaction mixture, stirring was maintained at a constant temperature for 24 hours, and then the reaction was stopped. The collected emulsion is the desired microsphere. Unreacted IA and other reagents were removed by high-speed centrifugation, and the microspheres obtained by centrifugation were then redispersed in distilled water.
2) Preparing carboxylated fluorescent microspheres:
dispersing the microspheres prepared in the above into an aqueous solution, and adjusting the pH value of the mixed solution by adding a certain amount of triethylamine; the samarium ion solution was then slowly added with stirring and the system temperature was maintained at 45 ℃ throughout the reaction. After the reaction was completed, the microspheres were collected by high-speed centrifugation, washed several times with water and ethanol to remove unreacted samarium ions and other reagents (solvents), and then dried in vacuo at 50 ℃.
The above-prepared was dispersed in an ethanol solution, and then an aromatic carboxylic acid solution was slowly added with stirring, and the system temperature was maintained at 40 ℃ throughout the reaction. After the reaction was completed, the microspheres were collected by high-speed centrifugation, washed several times with water and ethanol to remove unreacted aromatic carboxylic acid and other reagents (solvents), and then dried in vacuo at 50 ℃.
Claims (1)
1. The fluorescent microsphere with the surface rich in carboxyl is characterized in that the surface of the microsphere is rich in carboxyl and is hybridized by rare earth coordination fluorescent molecules, and the particle size of the microsphere is 0.1-2 mu m;
the synthesis is carried out according to the following steps:
1) Synthesizing high molecular microsphere with carboxyl-rich surface by soap-free emulsion copolymerization reaction: dissolving a polymer monomer for polymerization and olefine acid in methanol, taking sodium p-styrenesulfonate as a copolymerization emulsifier, adding an initiator, reacting under the protection of nitrogen, centrifuging, washing, and then dispersing in distilled water again;
2) Preparing fluorescent microspheres with carboxyl-rich surfaces: regulating the pH value of the mixed solution in the prepared high polymer microsphere aqueous dispersion liquid with the surface rich in carboxyl; then adding rare earth III ion solution for reaction, centrifuging, washing and vacuum drying after the reaction is finished; dispersing the obtained dry microspheres in ethanol, adding an organic ligand solution for reaction, centrifuging, washing and vacuum drying after the reaction is finished to obtain fluorescent microspheres with surfaces rich in carboxyl groups;
the polymer monomer for polymerization is at least one selected from styrene, substituted styrene, methacrylate and acrylic esters;
the olefine acid is at least one selected from itaconic acid, fumaric acid, itaconic anhydride, fumaric anhydride, unsaturated di-or poly-carboxylic acid and corresponding anhydride;
doping polyvinylpyrrolidone into the copolymerization emulsifier;
further, the polyvinylpyrrolidone is at least one of PVP K-30, PVP 360 and PVP 106;
the initiator for polymerization is at least one selected from potassium persulfate, azodiisobutyronitrile and benzoyl peroxide;
in the step 1), the dosage of the olefine acid is 0.5-10% of that of the polymerized monomer, the dosage of the copolymerized emulsifier is 0.2-2% of that of the polymerized monomer, and the dosage of the initiator is 1-5% of that of the polymerized monomer according to mass percent;
the reaction temperature is 40-90 ℃ under the protection of nitrogen, and the reaction time is 12-48 hours;
in the step 2), the amine used for regulating the pH value of the mixed solution is at least one selected from triethylamine and ethylenediamine, and the rare earth is at least one selected from terbium, europium, samarium, dysprosium and gadolinium; the pH value is 5-10, the reaction temperature is 30-85 ℃, and the reaction time is 3-48 hours;
in the step 2), the organic ligand is selected from at least one of 2,2' -bipyridine, 1, 10-phenanthroline, 8-hydroxyquinoline, indole, beta-diketone, aromatic carboxylic acid, triphenylphosphine oxide, dialkyl sulfoxide, pyridine nitrogen oxide, quinoline nitrogen oxide, macrocyclic polyether, macrocyclic polyketone, porphyrin, phthalocyanine, polyene compound and derivatives thereof;
in the step 2), the amount of amine is 0.5-10% of carboxylated microspheres, the amount of rare earth III ions is 0.5-5% of carboxylated microspheres, the amount of organic ligand is 0.5-10% of carboxylated microspheres, and the balance is ethanol and water.
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| CN111574652B (en) * | 2019-02-18 | 2021-08-17 | 中国科学院化学研究所 | Monodisperse carboxyl functional polymer microsphere based on redox reaction initiation, and preparation method and application thereof |
| CN113861430A (en) * | 2020-06-15 | 2021-12-31 | 中国科学院福建物质结构研究所 | Rare earth biological organic coordination nanoprobe synthesized by reverse microemulsion method and synthesis method |
| CN116218513A (en) * | 2023-03-03 | 2023-06-06 | 杭州谱康医学科技有限公司 | FITC-labeled fluorescent microsphere, preparation method and application |
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|---|---|---|---|---|
| CN1278534A (en) * | 2000-06-13 | 2001-01-03 | 复旦大学 | Polymer microball with fluorescent mark and its preparation |
| CN101392172A (en) * | 2008-11-01 | 2009-03-25 | 厦门大学 | Carboxylic fluorescent encoding microsphere and synthetic method thereof |
| CN105623651A (en) * | 2016-03-07 | 2016-06-01 | 江苏医诺万细胞诊疗有限公司 | Composite microsphere marker for conducting fluorescent marking on rear earth and preparation method of composite microsphere marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1278534A (en) * | 2000-06-13 | 2001-01-03 | 复旦大学 | Polymer microball with fluorescent mark and its preparation |
| CN101392172A (en) * | 2008-11-01 | 2009-03-25 | 厦门大学 | Carboxylic fluorescent encoding microsphere and synthetic method thereof |
| CN105623651A (en) * | 2016-03-07 | 2016-06-01 | 江苏医诺万细胞诊疗有限公司 | Composite microsphere marker for conducting fluorescent marking on rear earth and preparation method of composite microsphere marker |
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